The field of this invention is the transport of nucleic acids and more specifically the transport of oligonucleotides across biological membranes. Penetration of such molecules is hampered by their very hydrophilic and charged nature and efforts have been made to reduce the hydrophilic nature of such molecules by various means. Chemical modification of the internucleoside linkage can eliminate the charged character of the phosphodiester bond, e.g. by using methylphosphonates (Miller and Ts'o 1981, Annu Rep Med Chem 23:295) or can reduce it through incorporation of phosphorothioate bonds (Eckstein 1989, Trends Biochem Sci 14:97) or phosphorodithioate bonds (Nielsen 1988, Tetrahedron Lett 29:2911). Rudolph et al. (1996 in Nucleosides and Nucleotides 15:1725) introduced phosphonoacetate derivatives of oligonucleotides and Dellinger in U.S. Pat. No. 6,693,187 and its continuations U.S. Pat. No. 7,067,641; US2004/0116687 and US2006/0293511 present further data on the synthesis of such compounds. Phosphonoacetates were profiled as derivatives of oligonucleotides with reduced internucleoside charge that are highly nuclease resistant and, when designed as single stranded oligodeoxynucleotides, facilitate catalytic action of RNAseH upon binding to a complementary strand of RNA (in Sheehan et al, Nucl Acid Res 2003, 31:4109-4118). The thymidine dimers presented there display a decreased hydrophilicity at low pH; however, the cellular uptake of an oligonucleotide remained unchanged. In fact, cellular penetration was only achieved after elimination of the carboxylate charge group by esterification with methyl- or butyl groups.
In still other cases, lipophilic conjugation has been used to improve the cellular uptake of oligonucleotides such as single stranded oligodeoxynucleotides or double stranded siRNA molecules (Letsinger et al. in U.S. Pat. No. 4,958,013 or Proc. Natl. Acad. Sci., 86, 6553-6556, 1989 or by Manoharan et al. in U.S. Pat. No. 6,153,737 and U.S. Pat. No. 6,753,423 in combination with single stranded oligonucleotides; Soutschek et al. (2004) Nature, 432(7014), 173-178 or Wolfrum et al. (2007) in Nat Biotech 25:1149-1157 for the delivery of siRNA.
Very recently, Panzner in PCT/EP2007/011188 described nucleosides, nucleotides and nucleic acids derived thereof that are designed for improved cellular uptake and comprise one or more transfection enhancer elements, TEE's. The content of this PCT/EP2007/011188 is included herein by reference.
In brief, pH-responsive transfection enhancer elements (TEE's) have the general structure (I)Hydrophobic element-pH-responsive hydrophilic elements  (I)
The position of the hydrophilic element within the TEE structure may vary and PCT/EP2007/011188 teaches that the hydrophilic element can be located distal from the link between molecule and TEE. PCT/EP2007/011188 also mentions that the hydrophilic element can be located central within the TEE.
PCT/EP2007/011188 describes the pH-responsive hydrophilic element as weak acids having a pKa of between 2 and 6, preferred of between 3 and 5. Said weak acids may be selected from carboxyl groups, barbituric acid and derivatives thereof, xanthine and derivatives thereof, wherein in some embodiments the xanthine derivatives are pyrimidines.
PCT/EP2007/011188 also describes the pH-responsive hydrophilic element as zwitterionic structures comprising a combination of weak or strong acidic groups with weak bases, the latter having a pka of between 3 and 8, preferred of between 4.5 and 7.
PCT/EP2007/011188 further gives guidance how to achieve the specific pKa's of said hydrophilic elements, inter alia by substitution hydroxymethyl-, hydroxyethyl-, methoxymethyl-, methoxyethyl-, ethoxymethyl-, ethoxyethyl-, thiomethyl-, thioethyl-, methylthiomethyl-, methylthioethyl-, ethylthiomethyl-, ethylthioethyl-, chlorid-, chlormethyl-vinyl-, phenyl-, benzyl-, methyl-, ethyl-, propyl-, isopropyl- and tert-butyl or cyclohexyl groups.
The hydrophobic element of the TEE of PCT/EP2007/011188 can be linear, branched or cyclic chains with a minimum chain length of 6 units, sometimes as short as 4 units. The hydrophobic element often comprises more than 6 and up to 40 units, often between 6 and 20 units, wherein said units of said hydrophobic element often are carbon atoms, hydrocarbons or methylene groups.
PCT/EP2007/011188 also teaches that branching of the main chain of said hydrophobic element is possible and such branches may comprise building blocks, such as methyl-, ethyl-, propyl-, isopropyl-, methoxy-, ethoxy-, methoxymethyl-, ethoxymethyl-, methoxyethyl-, ethoxyethyl- and vinyl- or halogen groups or mixtures thereof.
In some embodiments of PCT/EP2007/011188 the hydrophobic element may derive from sterols, said sterols may be further substituted.
PCT/EP2007/011188 also mentions the insertion of one or more heteroatoms or chemical groups into the hydrophobic element of the pH-responsive transfection enhancer elements (TEE's). Such heteroatoms or chemical groups may be selected from —O—, —S—, —N(H)C(O)—, —C(O)O—, —OC(O)N(H)—, —C(O)—, —C(O)—N(H)—, —N(H)—C(O)—O—, —CH═N—, —O—C(O)—, —N═CH— and/or —S—S—, amino acids or derivatives thereof, α-hydroxyacids or β-hydroxy acids.
One central disclosure of PCT/EP2007/011188 is the hydrophilic-hydrophobic transition of TEE's in response to an acidification of the environment and application of such knowledge towards the design of nucleosides and nucleotides and detailed information on the design of modified nucleosides, nucleotides, internucleoside linkages or nucleic acids with enhanced membrane permeability is given therein.
As further described in the PCT/EP/2007/011188, the nucleobases contribute to the log D of a nucleic acid; their average log D at pH 7.4 is about −1.3 for DNA and −1.4 for RNA; the respective values at pH4 are −1.7 and −1.8 for DNA or RNA. The nucleobases therefore contribute a pH-dependent value of log D to the entire structure.
PCT/EP2007/011188 is also disclosing contributions of an average unit of the backbone, said contributions are −2.5 and −3 per abasic nucleotide in phosphodiester DNA and RNA, respectively, and −2.0 and −2.4 for the phosphorothioate building blocks.
The table 1 below integrates these values and provides a survey for the log D values of abasic polynucleotides and nucleic acids with average base use. The “monomer increment” describes the log D contribution for each additional nucleotide in a nucleic acid structure, the offset is the extrapolated log D for 0 nucleotides and the log D of larger structures is calculated as log D(n-mer)=offset+n*monomer increment, wherein n represents the number of monomer units in a nucleic acid.
Table 1: log D values for nucleic acid structures. The table shows calculated log D values for monomers to tetramers of abasic nucleic acids and the resulting monomer increment and offset values from these values. For the calculation of log D values of statistical 20 mer oligonucleotides, the contribution of average nucleobases was also taken into account.
oligomer withabasic oligomersnucleobases# of monomersmonomer1234incrementoffset20deoxypH 7.4−5.6−8.1−10.6−13.2−2.5−3.0−79.2pH 4−4.6−7.9−10.5−13.0−2.5−2.9−86.82′ OHpH 7.4−5.8−8.9−11.9−15.0−3.0−2.8−83.2pH 4−5.0−8.8−11.9−14.9−3.0−2.8−90.8PTO/DNApH 7.4−5.3−7.2−9.1−11.1−1.9−3.3−74.3pH 4−3.6−6.9−9.1−11.1−2.0−3.0−82.6PTO/RNApH 7.4−5.2−7.7−10.1−12.5−2.4−2.8−78.4pH 4−4.4−7.6−10.1−12.5−2.4−2.8−86.0
According to these calculations, oligonucleotides and longer nucleic acids are highly polar structures with log D values between −75 and −90 for average 20mers.
Also, the nucleic acids become even more polar at lower values of pH; this is a contribution of the nucleobases, not the backbone.